Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 August 2000 to 07 May 2001.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

1
Chemical structure
Reference substance name:
-
EC Number:
437-450-6
EC Name:
-
Cas Number:
64654-05-3
Molecular formula:
Hill formula: C28 H37 N
IUPAC Name:
N-(dodecylphenyl)naphthalen-1-amine
Test material form:
liquid: viscous
Details on test material:
Sponsor's identification: APAN
Description: redbrown viscous liquid
Lot number: EL01010B01/KZ8911.5
Storage conditions: room temperature in the dark
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for eight days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 127 to 169g, and the females weighed 1 16 to 158g and were approximately five to seven weeks old.
The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose of integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks (B & K Universal Ltd., Hull, UK).
The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity were controlled to remain within target ranges of 21 ± 2 °C and 55 ± 15% respectively. On one occasion the relative humidity was outside the respective target but was considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using random letter tables and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test material was administered daily, for twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 2 ml/kg/day of Arachis oil BP.
Vehicle:
arachis oil
Details on oral exposure:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in Arachis oil BP.
Formulations were prepared weekly and stored at approximately +4 °C in the dark.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Summary
The concentration of APAN in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Samples
The test material formulations were extracted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/ml.

Standards
Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.

Procedure
The standard and sample solutions were analysed by HPLC using the following conditions:
GC system: Hewlett-Packard 1050, incorporating autosampler and workstation
Column: Luna C18(250 x 4.6 mm id)
Mobile phase: acetonitrile:water (70:30 v/v)
Flow rate: 1.0 ml/min
UV detector wavelength: 217 nm
Injection volume: 10 μl
Retention time: ~ 13 mins

Homogeneity Determinations
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking in between sampling. Sampling was performed in triplicate.

Stability Determinations
The test material formulations were sampled and analysed initially and then after storage at approximately +4 °C in the dark for fourteen days.

Verification of Test Material Formulation Concentrations
The test material formulations were sampled and analysed within three days of preparation.

Linearity
A range of standard solutions were prepared covering the concentration range 0 to 0.2 mg/ml and analysed.
The detector response was shown to be linear up to 0.2 mg/ml.

Specificity
The diluent solvent acetonitrile and a blank Arachis oil BP(control) were analysed.
Analysis of the solvent and a blank Arachis oil BP (control) produced no signal that interfered with the signal due to the test material.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per dose (5 male/5 female)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen based on the results of the range-finding study. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.
Positive control:
Not required for this study.

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends. All observations were recorded.

Functional Observations
Prior to the start of treatment and on Days 5, 13, 19 and 26 all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during the final week of the study, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait; Tremors; Twitches; Convulsions; Bizarre/Abnormal/Stereotypic behaviour; Salivation; Pilo-erection; Exophthalmia; Lachrymation; Hyper/Hypothermia; Skin colour; Respiration; Palpebral closure; Urination; Defecation; Transfer arousal; Tail elevation

Functional Performance Tests
Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/hindlimb Grip Strength: An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response; Vocalisation; Toe pinch; Tail pinch; Finger approach; Touch escape; Pupil reflex; Startle reflex; Blink reflex

Bodyweight
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices: mean corpuscular haemoglobin (MCH); mean corpuscular volume (MCV); mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count: neutrophils (Neut); lymphocytes (Lymph); monocytes (Mono); eosinophils (Eos); basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic): Cresyl blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by 'Hepato Quick' and Activated partial thromboplastin time (APTT) was assessed by 'Preci Clot' using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea; Glucose; Total protein (Tot.Prot.); Albumin; Albumin/Globulin (AIG) ratio (by calculation); Sodium (Na+); Potassium (K+); Chloride (Cl-); Calcium (Ca++); Inorganic phosophorus (P); Aspartate aminotransferase (ASAT); Alanine aminotransferase (ALAT); Alkaline phosphatase (AP); Creatinine (Creat); Total cholesterol (Chol); Total bilirubin (Bili).
Sacrifice and pathology:
Pathology
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal, 60 mglml; Rhone MCrieux, Dagenharn, Essex, UK) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals; Brain; Epididymides; Heart; Kidneys; Liver; Ovaries; Spleen; Testes; Thymus

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Adrenals; Aorta (thoracic); Bone & bone marrow (femur including stifle joint); Bone & bone marrow (sternum); Brain (including cerebrum, cerebellum and pons); Caecum; Colon; Duodenum; Epididymides; Eyes; Gross lesions; Heart; Ileum; Oesophagus; Ovaries; Pancreas; Pituitary; Prostate; Rectum; Salivary glands (submaxillary); Sciatic nerve; Seminal vesicles; Skin (hind limb); Spinal cord (cervical); Spleen; Stomach; Jejunum; Kidneys; Liver; Lungs (with bronchi); Lymph nodes (cervical and mesenteric); Muscle (skeletal); Testes; Thymus; Thyroid/parathyroid; Trachea; Urinary bladder; Uterus
All tissues were despatched Precision Histology International, One Eyed Lane, Weybread, Diss,
Norfolk, UK for processing. The tissues from all control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with haematoxylin and eosin for subsequent microscopic examination. The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.
Since there were indications of treatment-related hepatic changes, examination was subsequently extended to include similarly prepared sections of liver from all animals in the other treatment groups.
Other examinations:
Not further examinations specified in the study report.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous painvise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.
The haematology variable basophils was not analysed since consistently greater than 30% of the data were recorded as the same value.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation was detected immediately before or up to ten minutes after dosing for animals of either sex treated with 1000 mg/kg/day from Day 4 onwards. Females from this treatment group developed hunched posture from Day 9 together with isolated incidents of prolonged increased salivation or fur staining. Pink staining on the cage tray-liners was detected for animals of either sex treated with 1000 or 150 mg/kg/day throughout the treatment period but this was probably normal excretion of the test material or its metabolite(s).
No treatment-related effects were detected at 15 mg/kg/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant reduction in bodyweight gain was detected for females treated with 1000 mg/kg/day during the first two weeks of the study. Females treated with 150 mg/kg/day also showed a slight reduction in bodyweight gain but this was confined to Week 2 only.
No adverse effect on bodyweight development was detected for the male treatment groups or for females treated with 15 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A reduction in dietary intake and food efficiency was detected for females treated with 1000 mg/kg/day during the first two weeks of the study. Food efficiency was slightly reduced for 150 mg/kg/day females but this was confined to Week 2 only.
No such effects were detected for the male treatment groups or for animals of either sex treated with 15 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slight reductions in haemoglobin, erythrocyte count and haematocrit were detected for animals of either sex at 1000 mg/kg/day but statistical significance was not achieved.
No such effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex from all treatment groups showed an increase in plasma bilirubin, increasing in a dose-related response.
No other treatment-related effects were detected.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Open-field assessment confirmed the clinical sign of hunched posture seen in 1000 mg/kg/day females from Week 2 onwards and increased salivation was detected in one male during Week 4.
No treatment-related effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 1000 mg/kg/day showed a statistically significant increase in liver weight, relative to bodyweight, compared with controls. Relative liver weight was also elevated for animals of either sex treated with 150 mg/kg/day but statistical significance was not achieved. Females treated with 1000 mg/kg/day showed an elevated relative spleen weight whilst absolute and relative adrenal weight was increased for this sex at both 1000 and 150 mg/kg/day.
No treatment-related organ weight changes were detected at 15 mg/kg/day.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological evaluation revealed treatment-related liver changes.
Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 1000 or 150 mg/kg/day, the effect extending to three 15 mg/kg/day males.
Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, it is generally considered to be adaptive in nature.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
The administration of APAN by oral gavage for a period of up to twenty-eight consecutive days resulted in treatment-related effects at dose levels of 1000, 150 and 15 mg/kg/day. Animals treated with 1000 mg/kg/day developed clinically observable signs during the study period involving increased salivation and hunched posture. Bodyweight development and dietary intake were adversely affected at this dose level during Weeks 1 and 2 but recovered over the final two weeks of treatment. A slight reduction in bodyweight and food efficiency was also detected at 150 mg/kg/day but this was confined to the females during Week 2 only. Haematological investigations revealed evidence of a mild anaemia in animals of either sex treated with 1000 mg/kg/day. A slight increase in relative spleen weight was also detected which probably indicates that a compensatory haemopoietic response has been initiated. In the absence of any histopathological correlates, the condition was considered to be minimal, representing a secondary response to treatment. Blood chemical investigations revealed a substantial increase in plasma bilirubin at all dose levels with many values outside normal ranges. This may have been associated with the anaemia present at the high dose but is more likely to be associated with liver change. Liver weights were elevated in animals of either sex treated with 1000 or 150 mg/kg/day and microscopic examination of liver sections revealed centrilobular hepatocyte enlargement at these dose levels and for three males treated with 15 mg/kg/day. Increased hepatic activity of this type is considered a normal adaptive biological response to xenobiotics and is usually the result of detoxification mechanisms involving hepatic enzyme induction. Nevertheless the unusually high levels of plasma bilirubin cannot be disregarded and, as such, a toxicologically significant liver effect is likely to have been demonstrated, albeit minimal at the lower dose levels.
The remaining organ weight changes detected were confined to a statistically significant increase in absolute and relative adrenal weight for 1000 and 150 mg/kg/day females. This increase may possibly have been a stress response associated with expansion of the adrenal cortex, but, in the absence of pathological changes, may be of no consequence.

Effect levels

open allclose all
Key result
Dose descriptor:
other: EC50
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks on result:
not determinable

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Summary Incidence if Daily Clinical Observations – Males

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 1

Day: 2

Day: 3

Day: 4

Day: 5

Day: 6

Day: 7

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

Generalised fur loss

No abnormalities detected

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

1

4

1

4

1

4

1

4

1

4

1

4

1

4

1

4

1

4

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 8

Day: 9

Day: 10

Day: 11

Day: 12

Day: 13

Day: 14

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

Generalised fur loss

No abnormalities detected

1

4

1

4

1

4

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

Increased salivation

No abnormalities detected

0

5

0

5

0

5

2

3

0

5

0

5

1

4

0

5

1

4

0

5

1

4

0

5

0

5

2

3

0

5

0

5

2

3

0

5

0

5

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 15

Day: 16

Day: 17

Day: 18

Day: 19

Day: 20

Day: 21

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

Increased salivation

No abnormalities detected

1

4

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

0

5

1

4

0

5

0

5

1

4

0

5

0

5

1

4

0

5

0

5

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 22

Day: 23

Day: 24

Day: 25

Day: 26

Day: 27

Day: 28

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

Increased salivation

No abnormalities detected

2

3

0

5

0

5

2

3

0

5

0

5

0

5

0

5

0

5

0

5

0

5

1

4

1

4

0

5

0

5

0

5

0

5

0

5

0

5

Pre = immediately before dosing

1h = one hour after dosing

5h = five hours after dosing

● = five hour observation not performed at weekend

# = increased salivation detected up to ten minutes after dosing – Days 4 to 28 inclusive

* = pink staining detected on cage tray-liners – Days 1 to 28 inclusive

 

Summary Incidence if Daily Clinical Observations – Females

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 1

Day: 2

Day: 3

Day: 4

Day: 5

Day: 6

Day: 7

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 8

Day: 9

Day: 10

Day: 11

Day: 12

Day: 13

Day: 14

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

Hunched posture

Increased salivation

No abnormalities detected

0

1

4

0

0

5

0

0

5

3

1

2

3

0

2

3

0

2

3

1

2

3

0

2

4

2

1

4

0

1

4

1

1

4

0

1

4

0

1

5

2

0

5

0

0

5

0

0

5

2

0

5

0

0

5

0

0

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 15

Day: 16

Day: 17

Day: 18

Day: 19

Day: 20

Day: 21

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

Hunched posture

Increased salivation

No abnormalities detected

5

0

0

5

0

0

5

0

0

5

2

0

5

0

0

5

0

0

4

0

1

4

0

1

4

0

1

4

0

1

4

2

1

4

0

1

5

0

0

5

1

0

5

0

0

5

0

0

5

0

0

5

0

0

5

0

0

Dose Level

(mg/kg/day)

Number of Animals

Clinical Observations

Number Showing Effects At Observation

Day: 22

Day: 23

Day: 24

Day: 25

Day: 26

Day: 27

Day: 28

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

Pre

1h

5h

0 (Control)

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

15

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

150 *

5

No abnormalities detected

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

1000 #*

5

Hunched posture

Increased salivation

No abnormalities detected

5

2

0

5

0

0

5

0

0

5

4

0

5

1

0

5

0

0

5

1

0

5

0

0

5

0

0

5

0

0

5

0

0

5

1

0

5

0

1

5

0

1

5

0

1

5

0

0

5

0

0

5

0

1

5

0

1

Pre = immediately before dosing

1h = one hour after dosing

5h = five hours after dosing

● = five hour observation not performed at weekend

# = increased salivation detected up to ten minutes after dosing – Days 4 to 28 inclusive

* = pink staining detected on cage tray-liners – Days 1 to 28 inclusive

 

Summary Incidence of Behavioural Assessments – Males

Pre-test

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

6

0

1

4

6

0

4

0

1

4

Urination

5

0

0

4

1

0

0

5

0

3

2

0

Transfer arousal

0

4

1

0

0

4

1

0

5

0

0

5

Week 1

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

0

1

4

0

4

0

1

4

Urination

5

0

5

0

0

5

0

4

1

0

Defecation

5

0

3

2

0

5

0

5

0

0

Transfer arousal

0

5

0

0

5

0

5

0

0

5

Week 2

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

1

4

0

1

4

0

4

0

1

4

Urination

4

1

0

4

1

0

5

0

4

1

0

Defecation

5

0

0

4

1

0

5

0

5

0

0

Transfer arousal

0

0

5

0

0

5

0

5

0

0

5

Week 3

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

1

4

0

1

4

0

1

4

0

1

4

Urination

3

2

0

4

1

0

3

2

0

4

1

0

Defecation

4

1

0

5

0

0

5

0

0

5

0

0

Transfer arousal

0

0

5

0

0

5

0

0

5

0

0

5

Week 4

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

0

4

0

1

4

0

2

4

Salivation

5

0

5

0

5

0

0

4

1

0

Urination

5

0

5

0

4

1

0

5

0

0

Defecation

5

0

5

0

4

1

0

5

0

0

Transfer arousal

0

5

0

5

0

0

5

0

0

5

 

Summary Incidence of Behavioural Assessments – Females

Pre-test

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

6

0

1

4

6

0

4

0

1

4

Urination

5

0

0

3

2

0

0

5

0

2

3

0

Transfer arousal

0

3

2

0

0

4

1

0

5

0

0

5

Week 1

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

0

4

0

4

0

4

Transfer arousal

0

5

0

5

0

5

0

5

Week 2

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

0

4

0

1

4

0

1

4

H

Bizarre behaviour

5

0

5

0

4

0

0

0

0

0

5

Urination

5

0

5

0

5

0

0

4

1

0

0

Defecation

5

0

5

0

4

1

0

5

0

0

0

Transfer arousal

0

5

0

5

0

0

5

0

0

5

0

H = Hunched posture

Week 3

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

0

4

0

4

0

1

4

H

Bizarre behaviour

5

0

5

0

5

0

0

0

0

5

Urination

5

0

5

0

5

0

5

0

0

0

Defecation

5

0

5

0

5

0

4

1

0

0

Transfer arousal

0

5

0

5

0

5

0

0

5

0

H = Hunched posture

Week 4

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

4

0

4

0

4

0

4

H

Bizarre behaviour

5

0

5

0

5

0

0

0

5

Transfer arousal

0

5

0

5

0

5

0

5

0

H = Hunched posture

 

Group Mean Functional Performance Test Values and Standard Deviations (SD) - Males

Dose Level

(mg/kg/day)

Number of Animals

 

Group Strength (g)

Motor Activity

Overall

Final 20% if Trail

Forelimb

Hindlimb

% Activity

% Mobile Activity

% Activity

% Mobile Activity

0 (Control)

5

mean

sd

755

249

340

84

28.3

5.6

5.0

1.0

15.9

8.0

1.9

0.8

15

5

mean

sd

707

188

339

83

27.0

4.2

5.1

0.3

9.5

3.2

1.2

0.6

150

5

mean

sd

700

160

318

85

25.6

3.1

4.5

0.6

14.5

2.6

2.0

0.5

1000

5

mean

sd

613

191

319

102

29.1

4.4

5.6

1.3

11.0

6.2

1.5

1.2

 

Group Mean Functional Performance Test Values and Standard Deviations (SD) - Females

Dose Level

(mg/kg/day)

Number of Animals

 

Group Strength (g)

Motor Activity

Overall

Final 20% if Trail

Forelimb

Hindlimb

% Activity

% Mobile Activity

% Activity

% Mobile Activity

0 (Control)

5

mean

sd

659

169

282

63

26.2

5.3

5.5

0.9

12.3

6.1

2.3

1.1

15

5

mean

sd

757

192

320

80

33.7

9.2

7.1

2.4

14.4

9.4

3.0

2.9

150

5

mean

sd

682

172

281

70

29.0

4.3

6.2

1.7

11.0

4.7

2.1

1.6

1000

5

mean

sd

629

226

301

63

30.4

1.9

6.5

0.9

12.9

3.1

2.1

0.8

 

Sensory Reactivity Assessments – Males

Summary Incidence

Dose Level (mg/kg/day)

0 (Control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

1

2

4

6

0

2

4

6

0

2

4

6

0

2

4

6

Grasp response

4

0

1

0

0

5

0

0

0

5

0

0

0

4

1

0

0

Vocalisation

4

1

0

0

0

5

0

0

0

5

0

0

0

4

1

0

0

Toe pinch

0

0

2

0

3

0

0

0

5

0

0

0

5

0

0

0

5

Tail pinch

0

0

1

4

0

0

0

5

0

0

0

5

0

0

0

5

0

Finger approach

0

0

5

0

0

0

4

0

1

0

5

0

0

0

5

0

0

Touch escape

0

0

0

5

0

0

0

5

0

0

0

5

0

0

0

5

0

Pupil reflex

0

0

0

5

0

0

0

5

0

0

0

5

0

0

0

5

0

Blink reflex

0

0

0

5

0

0

0

5

0

0

0

5

0

0

0

5

0

 

Sensory Reactivity Assessments – Females

Summary Incidence

Dose Level (mg/kg/day)

0 (control)

15

150

1000

Number of Animals

5

5

5

5

Number Classified As Observation

0

1

2

4

6

0

2

4

6

0

2

3

4

5

6

0

2

3

4

6

Grasp response

2

0

3

0

0

2

3

0

0

3

1

0

0

0

1

4

1

0

0

0

Vocalisation

1

2

1

1

0

2

3

0

0

3

0

1

0

1

0

4

0

1

0

0

Toe pinch

0

0

0

0

5

0

0

0

5

0

0

0

0

0

5

0

0

0

0

5

Tail pinch

0

0

0

5

0

0

0

5

0

0

0

0

5

0

0

0

0

0

5

0

Finger approach

0

0

4

0

1

0

4

0

1

0

5

0

0

0

0

0

3

0

0

2

Touch escape

0

0

0

0

5

0

0

4

1

0

0

0

3

0

2

0

0

0

3

2

Pupil reflex

0

0

0

5

0

0

0

5

0

0

0

0

5

0

0

0

0

0

5

0

Blink reflex

0

0

0

5

0

0

0

5

0

0

0

0

5

0

0

0

0

0

5

0

Group Mean Weekly Bodyweights and Standard Deviations (SD) - Males

Dose Level

(mg/kg/day)

Number of Animals

 

Bodyweight (g) at Day

0

7

14

21

28

0 (Control)

5

mean

sd

151

14

209

20

265

24

315

30

349

36

15

5

mean

sd

146

8

202

10

259

10

307

10

341

9

150

5

mean

sd

151

13

213

17

270

22

322

29

356

38

1000

5

mean

sd

153

16

209

19

260

22

308

22

337

22

 

Group Mean Weekly Bodyweights and Standard Deviations (SD) - Females

Dose Level

(mg/kg/day)

Number of Animals

 

Bodyweight (g) at Day

0

7

14

21

28

0 (Control)

5

mean

sd

135

13

175

18

202

21

223

22

236

19

15

5

mean

sd

133

10

165

13

190

15

213

20

226

22

150

5

mean

sd

139

12

170

13

190

11

207

12

219

16

1000

5

mean

sd

137

12

163

7

177

5

193

6

205

7

 

Group Mean Weekly Bodyweight Gains and Standard Deviations (SD) - Males

Dose Level

(mg/kg/day)

Number of Animals

 

Increase in Bodyweight (g) during Week

1

2

3

4

0 (Control)

5

mean

sd

58

8

56

8

50

9

34

8

15

5

mean

sd

56

3

57

4

48

4

34

6

150

5

mean

sd

63

5

57

7

52

8

34

12

1000

5

mean

sd

56

3

51

6

48

6

29

3

 

Group Mean Weekly Bodyweight Gains and Standard Deviations (SD) - Females

Dose Level

(mg/kg/day)

Number of Animals

 

Increase in Bodyweight (g) during Week

1

2

3

4

0 (Control)

5

mean

sd

40

8

28

5

21

2

13

5

15

5

mean

sd

32

6

25

8

23

6

13

3

150

5

mean

sd

31

4

*20

4

17

3

12

4

1000

5

mean

sd

**25

6

**14

2

16

4

11

1

* significantly difference from control group p<0.05

** significantly different from control group p<0.01

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material, APAN, to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related effects at all dose levels.
A "No Observed Effect Level" (NOEL) has, therefore, not been achieved.
The treatment-related changes detected at 150 or 15 mg/kg/day were minimal and, in isolation, were considered not to represent serious damage to health, as defined by the criteria given in the EC labelling guide of Commission Directive 93/21/EEC.
Executive summary:

The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).

 

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley Crl:CD (SD) IGS BR strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study.

 

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results.

Mortality: There were no deaths during the study.

 

Clinical Observations: Increased salivation was detected immediately before or up to ten minutes after dosing for animals of either sex treated with 1000 mg/kg/day from Day 4 onwards. Females from this treatment group developed hunched posture from Day 9 together with isolated incidents of prolonged increased salivation or fur staining. Pink staining on the cage tray-liners was detected for animals of either sex treated with 1000 or 150 mg/kg/day throughout the treatment period but this was probably normal excretion of the test material or its metabolite(s).

No treatment-related effects were detected at 15 mg/kg/day.

 

Behavioural Assessment: Open-field assessment confirmed the clinical sign of hunched posture seen in 1000 mg/kg/day females from Week 2 onwards and increased salivation was detected in one male during Week 4.

No treatment-related effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.

 

Functional Performance Tests: No treatment-related effects were detected.

 

Sensory Reactivity Assessments: No treatment-related effects were detected.

 

Bodyweight: A statistically significant reduction in bodyweight gain was detected for females treated with 1000 mg/kg/day during the first two weeks of the study. Females treated with 150 mg/kg/day also showed a slight reduction in bodyweight gain but this was confined to Week 2 only.

No adverse effect on bodyweight development was detected for the male treatment groups or for females treated with 15 mg/kg/day.

 

Food Consumption: A reduction in dietary intake and food efficiency was detected for females treated with 1000 mg/kg/day during the first two weeks of the study. Food efficiency was slightly reduced for 150 mg/kg/day females but this was confined to Week 2 only.

No such effects were detected for the male treatment groups or for animals of either sex treated with 15 mg/kg/day.

 

Water Consumption: No intergroup differences were detected.

 

Haematology: Slight reductions in haemoglobin, erythrocyte count and haematocrit were detected for animals of either sex at 1000 mg/kg/day but statistical significance was not achieved.

No such effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.

 

Blood Chemistry: Animals of either sex from all treatment groups showed an increase in plasma bilirubin, increasing in a dose-related response.

No other treatment-related effects were detected.

 

Organ Weights: Animals of either sex treated with 1000 mg/kg/day showed a statistically significant increase in liver weight, relative to bodyweight, compared with controls. Relative liver weight was also elevated for animals of either sex treated with 150 mg/kg/day but statistical significance was not achieved. Females treated with 1000 mg/kg/day showed an elevated relative spleen weight whilst absolute and relative adrenal weight was increased for this sex at both 1000 and 150 mg/kg/day.

No treatment-related organ weight changes were detected at 15 mg/kg/day.

 

Necropsy: No treatment-related macroscopic abnormalities were detected.

 

Histopathology: Histopathological evaluation revealed treatment-related liver changes. Centrilobular hepatocyte enlargement was observed in relation to treatment for animals of either sex treated with 1000 or 150 mg/kg/day, the effect extending to three 15 mg/kg/day males.

Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, it is generally considered to be adaptive in nature.

 

Conclusion: Oral administration of the test material, APAN, to rats for a period of twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in treatment-related effects at all dose levels. A "No Observed Effect Level" (NOEL) has, therefore, not been achieved.

The treatment-related changes detected at 150 or 15 mg/kg/day were minimal and, in isolation, were considered not to represent serious damage to health, as defined by the criteria given in the EC labelling guide of Commission Directive 93/21/EEC.